The cap is a unique structure found at the 5′-end of viral and cellular eukaryotic mRNA (1). This cap is critical for both mRNA stability and binding to the ribosome during translation. mRNA capping is a co-transcriptional modification resulting from a series of three chemical reactions (2). The 5′-triphosphate of the mRNA is first converted to a diphosphate by an RNA triphosphatase. The second reaction is a transfer of a GMP moiety from GTP to the 5′-diphosphate RNA by the guanylyltransferase (capping enzyme) to yield G5′-ppp5′-N. In general, this reaction involves a covalent attachment of the a-phosphate of GTP to the e-NH2 group of a lysine residue to yield a phosphoramide (P—N) bond with the concomitant release of pyrophosphate. In a third reaction utilizing S-adenosyl-L-methionine as the methyl donor, the transferred guanosine moiety is methylated by a methyltransferase at its N7 position to yield 7MeG5′-ppp5′-N (cap 0 structure). In some instances, a second methyl transfer reaction methylates the 2′-OH of the first nucleotide 3′ to the triphosphate bridge to yield 7MeG5′-ppp5′-N2′0Me (cap 1 structure) and it is the case for the mRNA Dengue virus.
Many viruses replicate in the cytoplasm of their eukaryotic host. Since cellular RNA capping is localized in the nucleus, these viruses often encode their own capping enzymes while relying on the host translation machinery for gene expression. Although the physical organization of the capping apparatus has diverged in cellular and viral systems, eukaryotic cellular and DNA virus guanylyltransferases have been grouped into a superfamily of covalent nucleotidyltransferases on the basis of structural and mechanistic features (3).
The crystal structure of the DNA virus PBCV-1 (Chlorella virus) guanylyltransferase in complex with GTP has illuminated the structural and mechanistic determinants of guanylyl transfer in this enzyme family (4). Covalent attachment of GMP to the enzyme is a hallmark of guanylyltransferase activity in this family. Unlike DNA viruses, however, the classification and mechanism of RNA virus guanylyltransferases is elusive. Only a few guanylyltransferase activities from RNA viruses have been assigned to viral proteins because they do not share obvious amino acid sequence homology with covalent nucleotidyltransferases. The sole example of a structurally defined RNA virus guanylyltransferase is the crystal structure of the Reovirus core at 3.6 Å resolution comprising the λ2 subunit (5). However, it is a double strand-RNA virus, and not a single strand-RNA virus. As RNA capping is essential for several viruses (6), it is a potential target for antiviral design.
The guanosine analogue Ribavirin is a broad spectrum antiviral agent discovered about thirty years ago (7). Its mechanism of action has remained controversial (8). Like most nucleoside analogues, Ribavirin is phosphorylated at its 5′-position upon penetration into the cell. Ribavirin 5′-monophosphate is a potent inhibitor of the cellular enzyme inosine 5′-monophosphate dehydrogenase (IMP-DH). This inhibition results in depletion of the intracellular guanosine nucleotide pool which feeds capping and polymerase enzymes of both viral and cellular origin. Consequently, the Ribavirin-depressed guanosine nucleotide pool may exert an indirect antiviral effect because viral enzymes would not compete advantageously for guanosine nucleotide with cellular enzymes. In addition, Ribavirin nucleotides might have a viral target, such as RNA capping, responsible for the observed antiviral effect (reviewed in (8)), but direct evidence for this mechanism was lacking. Elucidation of the Ribavirin mechanism of action has been plagued by the possible involvement of both direct and indirect mechanisms.
Viruses from the Flaviviridae family are sensitive to Ribavirin (9, 10). The genus Flavivirus comprises important human pathogens such as West Nile, Dengue and Yellow Fever viruses. These mosquito-borne viruses are currently expanding their distribution over the world. West Nile virus introduction in North America may be an important milestone in the evolving history of this virus, as exemplified with recent outbreaks in the New-York area (11). The Camargue area in France has re-witnessed West Nile virus infection of horses after 40 years. Likewise, Dengue virus, an agent responsible for hemorrhagic fever, is infecting more than 50 million persons annually with an increasing incidence in tropical areas around the world.
The Flavivirus single-stranded RNA genome is of positive polarity, and capped with a cap 1 structure (12). The guanylyltransferase has not yet been identified. Structural insights into viral RNA capping and its inhibition may reveal a putative target for Ribavirin and help the identification and design of inhibitors directed against Flaviviruses. Once identified, such inhibitors will be useful in the treatment of diseases resulting from Flavivirus infection.
This invention discloses an isolated and purified polypeptide capable of acting as a guanylyltransferase and methyltransferase comprising capping enzyme of flavivirus (CEF). The polypeptide of the invention comprises a N-terminal module (subdomain 1), a SAM-binding core (subdomain 2), and a C-terminal sequence (subdomain 3) located between subdomains 1 and 2, and forming the bottom of a narrow cleft. The subdomain 1 of the polypeptide of the invention starts with a helix A1-turn-helix A2 motif. The subdomain 2 polypeptide of the invention is comprised of a twisted mixed β-sheet comprising 7 β-strands (β1 to β7) and 5 helices (α1 to α5) and its subdomain 3 is positively charged.
The polypeptide of the invention is designated “CEF” (Capping Enzyme of Flavivirus). This invention provides the general structure of CEF. The amino acid sequences of three CEFs—for the Dengue virus, the West Nile Virus, and Yellow Fever Virus—are also disclosed and these sequences are respectively represented by SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3 in the sequence listing in the appendix.
The invention relates to polypeptides homologous to a polypeptide whose amino acid sequence is represented by SEQ ID No. 1, SEQ ID No. 2 or SEQ ID No. 3. More particularly, the invention relates to polypeptides which have at least about 95% homology with amino acid sequences represented by SEQ ID No. 1, SEQ ID No. 2 or SEQ ID No. 3.
The invention also concerns a nucleic acid molecule comprising or constituted of an encoding nucleic sequence for a polypeptide capable of acting as a guanylyltransferase and methyltransferase comprising capping enzyme of flavivirus (CEF). The invention also concerns nucleotide sequences derived from the above sequences, for example, from the degeneracy of the genetic code, and which encode for proteins having characteristics and properties of CEF.
The invention includes polyclonal or monoclonal antibodies directed against a polypeptide of the invention, a derivative or a fragment thereof. These antibodies can be prepared by known methods. The antibodies are useful in identifying new CEF or the homologues of this enzyme in other virus belonging to the flavivirus genus.
The invention also concerns a vector comprising at least one molecule of the nucleic acid above, advantageously associated with adapted control sequences, together with a production or expression process in a cellular host of the CEF of the invention or a fragment thereof. The preparation of these vectors as well as the production or expression in a protein host of the invention can be carried out by molecular biology and genetic engineering techniques well known to the one skilled in the art.
An encoding nucleic acid molecule for a polypeptide capable of acting as a guanylyltransferase and methyltransferase comprising capping enzyme of flavivirus (CEF) or a vector according to the invention can also be used to transform animals and establish a line of transgenic animals. The vector used is chosen in function of the host into which it is to be transferred. It can be any vector such as a plasmid. Thus, the invention also relates to cellular hosts expressing a polypeptide capable of acting as a guanylyltransferase and methyltransferase comprising capping enzyme of flavivirus (CEF) obtained in conformity with the preceding processes.
The invention also relates to nucleic and oligonucleotide probes prepared from the molecules of nucleic acid according to the invention. These probes, marked advantageously, are useful for hybridisation detection of CEF in other viruses of the Flavivirus genus. According to prior art techniques, these probes are put into contact with a biological sample. Different hybridization techniques can be used, such as Dot-blot hybridisation or replica hybridisation (Southern technique) or other techniques (DNA chips). Such probes constitute the tools making it possible to detect similar sequences quickly in the encoding genes of different virus of the Flavivirus genus. The oligonucleotide probes are useful for PCR experiments, for example, in a diagnostic sense.
The invention can also be useful in methods for determining the inhibitory power of a biologically active compound acting as a competitive inhibitor of GTP comprising:                a) incubating CEF with radiolabeled GTP,        b) adding selected different concentrations of the biologically active compound,        c) assaying resulting radiolabeled CEF-GTP complex produced from the incubation,        d) quantifying an amount of radiolabeled CEF-GTP complex produced from the assay,        e) comparing the amount to a binding affinity constant of GTP to CEF, and        f) determining the inhibitory power of the biologically active compound.        
More particularly, the incubating step in the methods of the invention is conducted at about 52 μM of GTP. Selected different concentrations of GTP are about 0 μM, about 10 μM, about 20 μM, about 50 μM, about 100 μM, about 200 μM, about 300 μM, about 500 μM, and about 800 μM. Assaying in the methods of the invention comprises UV-crosslinking of α-32P-GTP to CEF.
The invention relates to methods for selecting an inhibitory biologically active compound capable of reducing CEF binding to GTP as an antiviral pharmaceutical agent comprising selecting biologically active compounds with a binding affinity higher than the binding affinity constant of GTP to CEF.
The biologically active compound which can be used in the methods of the invention can be a nucleoside, a nucleoside analogue or a non-nucleoside molecule, for example.
We discovered that the triphosphate form of acyclovir and a vectorized form of acyclovir 5′-monophosphate are good inhibitors of the CEF enzyme. Thus, the invention concerns the use of acyclovir 5′-triphosphate or a vectorized form of acyclovir 5′-monophosphate for preparing a medicine useful in treating or preventing diseases resulting from Flavivirus infection. The terms “vectorized form” relates to any vector capable of transporting acyclovir 5′-monophosphate to a particular cell such as an infected cell and to introduce acyclovir 5′-monophosphate into the cell. All kinds of vectors known by those skilled in the art can be used in the invention.